Elastases
Elastases are one of several proteolytic enzymes found in the pancreatic juice. Elastase is a member of the serine protease family, a family of proteolytic enzymes that includes other digestive enzymes, such as chymotrypsin and trypsin, and some of the proteases in the blood clotting and complement enzymatic cascades. Members of the serine protease family usually share about 40% homology with other members within the family.
The serine proteases are a class of proteolytic enzymes characterized by the presence of a uniquely reactive serine side chain. The reactive serine group forms a covalent ester bond to the carbonyl carbon atom of a susceptible bond of a polypeptide substrate to form an acyl-enzyme intermediate. Chemical and kinetic studies show that the chemical reactivity of the serine residue is a consequence of a charge relay system, consisting of a histidine side chain imidazole hydrogen bonded to a buried carboxylate of an aspartic acid. The aspartic acid provides the binding site for a proton which, in the transition state, is transferred between nucleophiles, i.e. between the reactive serine of the enzyme and the leaving group of the substrate. This relay system increases the nucleophilicity of the active site serine.
Serine proteases also are characterized by having their catalytically functional groups arranged in the same geometrical relationship. The enzyme binding template is made up of a number of elements acting together: an antiparallel beta-sheet site for binding the substrate polypeptide chain to be acylated, specific side chain binding sites that vary with the particular enzyme; a less specific leaving-group site; a site for hydrogen bonding to a tetrahedral oxyanion; and naturally the reactive serine side chain for covalent binding to a substrate's carbonyl carbon atom.
Polypeptide substrate amino acid residues extending from the cleaved bond toward the N-terminus are typically denoted as P1, P2, P3, and those extending from the cleaved bond toward the C-terminus denoted P'1, P'2, and P'3. The corresponding binding sites on the enzyme are denoted S1, S2, S3, and S'1, S'2, and S'3. Sites S1, S2 and S3 are part of the anti-parallel beta sheet and form part of the crevice for peptide binding. The other wall of the crevice is made up of other backbone peptide links. When a polypeptide substrate is bound at the binding site, the peptide side chains fit into a crevice on the enzyme surface.
Trypsin, the most specific of the serine proteases, attacks preferentially at peptide bonds following an arginine or a lysine at P1. Chymotrypsin rapidly hydrolyzes peptide bonds following an aromatic side chain at P1. Elastase does not display quite a marked specificity, but generally prefers an uncharged nonaromatic side chain especially alanine at the P1 position. In elastase, the crevice is partially occluded by the side chain of Val-216, and the bottom of the crevice is partly filled by the side chain of threonine-226, leaving room for binding of small P1 side chains only. Because of the small binding energy available from the interaction between the P1 side chain and the crevice surface in elastase, catalysis is more dependent upon enzyme substrate contacts remote from the cleaved bond.
Proteolytic Enzyme Secretion from the Pancreas
The pancreas consists in part of exocrine tissue. Acinar cells are derived from exocrine tissue and function in the secretion of numerous pancreatic enzymes which assist digestion in the gastrointestinal tract. The proteolytic enzymes secreted by the acinar cells include elastase, trypsin, chymotrypsin, and carboxypeptidase. Trypsin, chymotrypsin, and elastase split whole and partially-digested proteins into polypeptides of different sizes; then, carboxypeptidase breaks down the polypeptides into individual amino acids. The principal enzyme for digesting carbohydrates in the gut is pancreatic amylase. It hydrolyses starches, glycogen, and most other non-cellulosic carbohydrates to form disaccharides and trisaccharides. The main enzymes for fat digestion are pancreatic lipase, cholesterol esterase, and phospholipase. Pancreatic lipase hydrolyses neutral fat into fatty acids and monoglycerides. Cholesterol esterase hydrolyses cholesterol esters, and phospholipase removes fatty acid molecules from phospholipids.
Pancreatic cells secrete the proteolytic enzymes as inactive forms, such as trypsinogen, chymotrypsinogen, procarboxypeptidase, and proelastase. These enzymes are not activated until they reach the duodenum. Secretion of enterokinase from the intestinal mucosa is triggered by chyme entering the duodenum. Trypsinogen is activated by enterokinase or existing duodenal trypsin. Both chymotrypsinogen, procarboxypeptidase and proelastase are primarily activated by trypsin. Along with the inactive proteolytic enzymes, the acinar cells also secrete trypsin inhibitor. Trypsin inhibitor prevents the activation of the proteolytic enzymes inside acinar cells and pancreatic ducts.
Four molecules control proteolytic enzyme secretion from the pancreas: acetylcholine and the hormones, gastrin, cholecystokinin (CCK), and secretin. Acetylcholine, gastrin, and CCK all stimulate the acinar cells of the pancreas to secrete enzymes. These three molecules also stimulate the ductal cells to secrete sodium bicarbonate and water. Secretin, however, stimulates the secretion of large quantities of this sodium bicarbonate "solution" which serves to wash/move pancreatic enzymes through the various ducts into the duodenum.
Acetylcholine is released from the parasympathetic vagus nerve endings and other cholinergic nerves of the enteric nervous system. CCK and secretin are secreted by the duodenal and upper jejunal mucosa when chyme enters the small intestine. CCK occurs in several forms. The most common are the 8- and 33-amino acid forms. CCK molecules are released from cells of the duodenal and jejunal mucosa. Proteases and peptones from partial digestion of proteins and long chain fatty acids stimulate the release and transport of CCK. In the pancreas, CCK interacts with specific acinar cell-surface receptors. Via the phosphoinositol pathway, the CCK-receptor complex mobilizes intracellular Ca.sup.2+ and cyclic guanosine monophosphate (cGMP) which in turn activates protein kinase. Secretin has 27 amino acids and interacts with specific cell membrane receptors of the ductal cells. The receptors activate an intracellular cyclic adenine monophosphate (cAMP) system which stimulates cellular secretion.
Pancreatic secretion occurs in three different phases, the cephalic, gastric, and intestinal phases. During the cephalic phase, a nerve stimulates secretion in the stomach and release of acetylcholine from vagal nerve endings in the pancreas. Acetylcholine governs the release of moderate amounts of enzymes. Because very little water and sodium bicarbonate are secreted during this phase, only small amounts of enzyme are washed into the duodenum.
During the gastric phase, large amounts of gastrin formed in the stomach stimulate the pancreas to secrete more enzymes. Still, lack of water and sodium bicarbonate assure that very small quantities of enzymes reach the duodenum. It is during the intestinal phase, when chyme enters the duodenum, that secretin is produced and transported to the pancreas. There secretin stimulates the release of large quantities of sodium bicarbonate solution which wash the enzymes into the duodenum. At this time, the mechanisms for the active transport of sodium ions (Na.sup.+) and bicarbonate ions are not fully understood, but it has been suggested that active transport of sodium ions is accomplished by a Na.sup.+, H.sup.+ -ATPase at the apical membrane. CCK also stimulates increased pancreatic enzyme secretion during this last phase.
Inhibition during the cephalic phase involves the counteraction of parasympathetic innervation by sympathetic innervation. However, norepinephrine has no effect on basal pancreatic secretion. During the gastric phase, any factor which directly affects the retention of chyme in the stomach will prevent or reduce the secretion of CCK and secretin. If fat is introduced into the ileum during the intestinal phase, an unidentified hormonal factor has been shown to inhibit pancreatic secretion.
Excessive active serine protease production, including elastase production, may result in tissue wasting. For example, acute pancreatitis may result by the activation of proteolytic enzymes within the pancreas. A number of factors, such as endotoxins, exotoxins, viral infections, ischemia, anoxia and direct trauma, are believed to activate these proenzymes. The active enzymes then digest cellular membranes and cause edema, interstitial hemorrhage, vascular damage, coagulation necrosis, fat necrosis, and parenchymal cell necrosis. On the other hand, very low levels of serine protease production may indicate the destruction of acinar cells.